The overall goal of Project 2 of the Research Program is to define the essential role of the abnormal hyaluronan (HA)-based matrix that is synthesized by airway smooth muscle (ASM) and airway epithelial (AE) cells in response to pathological stimuli (viral, allergen) and that modulates asthmatic inflammation through interactions with inflammatory cells (monocytes/macrophages, mast cells, eosinophils). Our data show that cultures of ASM and AE cell cultures respond to poly(l:C) (a viral mimetic) and tunicamycin (endoplasmic reticulum (ER) stress) by synthesizing HA-based structures that are highly and selectively adhesive for binding non-activated monocytes as well as un-primed primary mast cells at 4 degrees C. At 37 degrees C, the adhesion of the monocytes is followed by a rapid (10-15 min) activation and phagocytosis of the HA matrix by a mechanism that includes capping of CD44, a HA-binding cell surface protein on the monocytes that is required for the phagocytic response. Additional strong, correlative histological data indicate that this process occurs in vivo in asthmatic individuals and in the aeroallergen/pathogen mouse model of asthma that is central to this Research Program. The same histological sections also show a strong ER stress response in AE cells while nearby vascular endothelial cells do not. The aims of this Project are to treat murine ASM cell cultures with poly(l:C) and determine: Aim 1) the mechanism by which HA is synthesized and incorporated into the HA-based structures;Aim 2) the mechanism by which leukocytes adhere to these structures;and Aim 3a) the mechanism by which monocytes cap and phagocytose the structures. Specific Aim 3b will determine how primary mast cells (un-primed and primed) respond to incubation at 37 degrees C after selective adhesion to the HA structures at 4 degrees C. Specific Aim 4 will develop a novel lift culture model for growing AE cells on a native basement membrane substrate under conditions that promote AE differentiation and determine the response of the differentiated cultures to poly(l:C) and tunicamycin. Transgenic mouse models (null in CD44, in L-selectin, in both, and in TSG-6) will be used in the in vitro culture models and in vivo in the murine aeroallergen/pathogen model to determine the contribution of these molecules in formation of the HA matrix and the subsequent leukocyte adhesion and activation responses. This Project interacts strongly with Project 1, which will determine leukocyte tyrosine nitration/oxidation parameters in poly(l:C) and tunicamycin treated AE cultures with and without adhered monocytes;and with Project 3, which will determine how eosinophils respond to interaction with the HA structures. The use of Cores B and C will provide human tissue, and Core D will provide mouse tissues and the aeroallergen/pathogen model in the null mice.